为探索一种适用于茶树蛋白质双向电泳的样品制备方法,研究比较了TCA/丙酮沉淀法、改良的Tris-HCl抽提法,以及酚-甲醇/醋酸铵沉淀法3种植物蛋白质样品制备方法。结果表明,改良的Tris-HCl抽提法较适合于茶树蛋白质双向电泳的样品制备。该改良方法制备的茶树蛋白质样品经双向电泳分离,可获得1117±9.89个蛋白质点,其中大部分蛋白质的等电点集中在pI5~7之间,相对分子质量集中在15.00~95.00kD之间,背景清晰且蛋白质得率较高,能满足茶树蛋白质组学研究的需要。
In order to develop an efficient protein extraction method suitable for two-dimensional electrophoresis from tea plant, three methods (TCA/acetone precipitation, improved Tris-HCl extraction and phenol-methanol/ammonium acetate precipitation) for total protein extraction in plant were compared. The result showed that the improved Tris-HCl extraction was the best method suitable for tea leaf protein sample preparation in these three techniques and the extracts were separated by two-dimensional electrophoresis. Consequently, 1117±9.89 protein spots were found and their isoelectric points were found to lie between pI5~7. The relative molecular weight of proteins was between 15.00~95.00kD. The improved Tris-HCl extraction could get the highest resolution of two dimensional electrophoresis and high yield of protein, and it could meet the requirement for the research on tea proteomecis.
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